Hyphenating Liquid Chromatography with MS in Proteomics

The rise of mass-spectrometry in proteomics research

Decades of significant advancements in mass-spectrometry technologies have resulted in better resolution, higher sensitivity and mass accuracy, simplification of calibration, and the standardization of acquisition parameters, which have enabled more users to access MS for day-to-day usage.

These major developments have been so large that gain in results has clearly been observed, even without improving front-end liquid chromatography (LC) systems! The number of identifiable peptides and proteins has increased by approximately 10 times, the achievable dynamic range has widened, and many laboratories across the world have adopted LC-MS.

What is the next step?

The logical move is to make proteomics a routine tool for precision medicine and translation research studies. By unleashing the power of unbiased protein detection and quantification, researchers can solve real-life problems like finding drug targets, biomarkers, etc.

The place of liquid-chromatography in LC-MS proteomics

The advanced application requirements revealed some bottlenecks in performing low-flow LC-MS proteomics experiments. These bottlenecks were linked to the need of expert technical skills and several years of hands-on experience, deep knowledge of technology, and troubleshooting skills required to operate high-end nanoLC-MS systems.

Consequently, the demand for better sample throughput and utilization of MS detectors, reduced overhead time, lower carryover for complex samples like biological fluids, higher separation performance, and robust 24/7 operation pushed forward the development of more user-friendly front-end low-flow systems.

The recent addition of the Thermo Scientific™ Vanquish™ Neo UHPLC system to the proteomics researchers’ toolbox shifts the paradigm of nano-, capillary- and micro-flow LC-MS proteomics usage by bringing closer high-performance LC-MS instruments and user needs.

What value does liquid chromatography add to LC-MS proteomics?

Number 1 is the separation of compounds in time. Despite the fact that high-resolution accurate-mass (HRAM) MS resolves several hundreds of individual compounds in one spectrum, the complexity of proteomics samples is significantly larger with several hundred thousand peptides in one sample. Thus, the separation of compounds in time is essential to reduce the sample complexity and allow mass-spectrometry to detect as many targets as possible.
Number 2 is the reduction of ion suppression. The presence of multiple species competing for ionization inhibits ionization efficiency and suppresses the signal for individual analytes. The improved separation of compounds allows one to achieve stronger signals and greater sensitivity.
Number 3 is the concentration of analytes from the sample on the column and elution in one narrow band. This improvement delivers a wider dynamic range and gives lower limits of detection and quantification.

Do you know other advantages of coupling LC with MS? Let us discuss them in the comments below.